1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing pyrroloquinoline quinine (PQQ) using a bacterium of the genus Methylobacterium or Hyphomicrobium which has been modified to enhance the expression of the pqq gene cluster and/or gene(s) encoding a precursor for PQQ biosynthesis.
2. Brief Description of the Related Art
Pyrroloquinoline quinine (PQQ) is the cofactor for several bacterial dehydrogenases including glucose dehydrogenase and methanol dehydrogenase located in the periplasm of Gram-negative bacteria and may be accumulated extracellularly by cultivating these bacteria. All of the PQQ-producing strains have PQQ-dependent quinoproteins.
A method for the preparation of PQQ using bacteria belonging to the genera Achromobacter, Methylobacillus, Methylomonas, Methanomonas, Protaminobacter, Methylobacterium, Protomonas, Mycoplana, Ancyclobacter, Microcyclus, Hyphomicrobium, Xanthobacter, Thiobacillus, Alteromonas, Methylophaga and some species of the genus Pseudomonas, which are cultivated in a medium containing methanol and/or methylamine as a carbon source, is disclosed (EP0206471 B1).
Genes involved in PQQ biosynthesis have been characterized for several bacteria, including Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens, and Gluconobacter oxydans. Six genes and seven genes are required in K. pneumoniae and M. extorquens (AM1), respectively, and only four genes are required in A. calcoaceticus for PQQ biosynthesis. The pqqA genes from different species encode small peptides of 23 to 29 amino acids which contain conserved glutamic acid and tyrosine residues. PQQ is derived from the two amino acids glutammic acid and tyrosine encoded in the precursor peptide PqqA. Presumably, five reactions are necessary to form PQQ (Puehringer et al. BMC Biochemistry 2008, 9:8 doi:10.1186/1471-2091-9-8). Some of the proteins involved in PQQ biosynthesis have been functionally characterized.
The PqqB protein is supposed to be involved in transport of PQQ into the periplasm. It has been reported that a knock out of PqqB produces small amounts of PQQ in the cytosol and that no PQQ is secreted into the periplasm (Velterop et al. Journal of bacteriology (1995) 177(17):5088-5098). The PqqC protein is an oxidase which catalyzes the final step in PQQ formation. The functions of PqqD protein are still unknown. Recently, the interaction of PqqD protein with the radical SAM enzyme PqqE has been demonstrated in K. pneumoniae (Wecksler S R et al. Chem Commun 2010 Oct. 7; 46(37):7031-3).
Based on sequence analysis and homology models, it is supposed that PqqE recognizes the PqqA protein and forms a bond between the C atoms from the glutammic acid and tyrosine in PqqA, and, therefore, enables recognition of the modified PqqA by PqqF protein. In the next stage, PqqF is suggested to catalyze cutting of the generated glutammic acid-tyrosine pair out of PqqA protein (Puehringer et al. BMC Biochemistry 2008, 9:8 doi:10.1186/1471-2091-9-8).
It was revealed that PQQ biosynthesis in Escherichia coli, which does not possess an ability to produce PQQ, can be achieved through the expression of pqq gene clusters of A. calcoaceticus (Goosen N. et al. J Bacteriol (1989) 171:447-455), K. pneumoniae (Meulenberg J J M et al. FEMS Microbiol Lett (1990) 71:337-344), and G. oxydans (Yang et al. Journal of Industrial Microbiology&Biotechnology (2010), 37(6), 575-580). Also, the positive effect of copies of some pqq genes on PQQ production in Methylobacterium extorquens AM1 was described (Wu, Bo; Zhao, Yong-fang; Wang, Yin-shan.Wuhan Daxue Xuebao, Ziran Kexueban (1999) 45(6), 869-872). Deletion of mxbM gene and pqqABC/DE gene cluster in M. extorquens AM1 led to absence of PQQ production. The deletion mutant transformed by a plasmid harboring mxbM gene and pqqABC/DE gene cluster produced PQQ in larger amounts than the wild-type, presumably as a result of the higher copy number of pqq genes. (Toyama H. and Lidstorm M E. Microbiology (1998), 144, 183-191).
But, currently, there have been no reports of enhancing expression of the pqq gene cluster in a bacterium, belonging to the genus Hyphomicrobium, and enhancing expression of the additional pqqA gene(s) encoding a precursor for PQQ biosynthesis in a bacterium of the genera Methylobacterium or Hyphomicrobium for the purpose of producing PQQ.